Janus kinase 2 (commonly called JAK2) is a human protein that has been implicated in signalling by members of the type II cytokine receptor family (e.g. interferon receptors), the GM-CSF receptor family (IL-3R, IL-5R and GM-CSF-R), the gp130 receptor family (e.g. IL-6R), and the single chain receptors (e.g. Epo-R, Tpo-R, GH-R, PRL-R). JAK2 signalling is activated downstream from the prolactin receptor. Mutations in JAK2 have been implicated in polycythemia vera, essential thrombocythemia, and other myeloproliferative disorders. This mutation, a change of valine to phenylalanine at the 617 position, appears to render hematopoietic cells more sensitive to growth factors such as erythropoietin and thrombopoietin.
Polycythemia vera (also known as erythremia, or primary polycythemia) is a blood disorder in which the bone marrow makes too many red blood cells. It may also result in the overproduction of white blood cells and platelets. Most of the health concerns associated with polycythemia vera are caused by the blood being thicker as a result of the increased red blood cells. It is more common in the elderly and may be symptomatic or asymptomatic.
It is of benefit to provide a rapid and simple test for diagnosis of polycythemia vera.
U.S. Pat. No. 7,429,456 describes the identification of the valine to phenylalanine (V617F) mutation in JAK2, and describes a test for detecting the presence of the mutation. SEQ ID NO 1 of U.S. Pat. No. 7,429,456 gives the amino acid sequence of the V617F form of human JAK2, and SEQ ID NO 2 of U.S. Pat. No. 7,429,456 gives the nucleic acid sequence encoding the amino acid sequence (the gene sequence mutation is referred to as G1849T); reference is also made to the wild type form of the protein, under NCBI accession number NM 004972. The diagnostic test described in this patent makes use of nucleic acid probes which span the mutated T nucleotide at position 1849 of the nucleic acid sequence. The presence of the mutation is detected by PCR amplification of the region flanking position 1849, followed by hybridisation with a probe including the T1849 nucleotide. If there is hybridisation, then the mutation is present. Alternatively, the PCR primers may be directed to the mutant sequence, such that the mutant form will be selectively amplified. The mRNA sequence of JAK2 (SEQ ID NO 4) is given in FIG. 1 of the present application. Position G1849 corresponds to nt 2343 of SEQ ID NO 4 of FIG. 1. Such assays are specific for the particular mutation.
It would be useful to provide an alternative diagnostic test.
Our co-pending patent application GB 1100150.0 describes a method for detecting and analysing SNPs in target genes, based on preferential amplification of the mutant sequence. The melting temperature (Tm) of double stranded DNA depends on the extent of base pair hybridisation; where there is a mismatch between a probe and a target sequence (for example, due to the presence of a SNP) then the Tm will differ compared with when there is no mismatch. The method described in GB 1100150.0 involves the use of PCR primers to flanking regions of the target sequence in combination with a blocking probe which preferentially hybridises to either the wild type or the mutant sequence. The probe is designed such that it has a lower Tm when binding to the sequence to be blocked (for example, the wild type sequence) than when binding to the sequence to be amplified (for example, the mutant sequence). Amplification is carried out at a temperature such that the probe hybridises to the sequence to be blocked but not the sequence to be amplified, and thus only the other sequence is amplified. The same probe may then be used to detect the presence of the amplified sequence, and to detect the relative ratios of the amplified to non-amplified sequence.
The present invention applies related methods to the detection of mutations in JAK2.